The present invention pertains to a chimeric adenovirus fiber protein comprising a constrained nonnative amino acid sequence.
Despite their prior poor reputation as major pathogenic agents that lead to numerous infectious diseases, adenoviruses (and particularly, replication-deficient adenoviruses) have more recently attracted considerable recognition as highly effective viral vectors for gene therapy. Adenoviral vectors offer exciting possibilities in this new realm of therapeutics based on their high efficiency of gene transfer, substantial carrying capacity, and ability to infect a wide range of cell types (Crystal, Science, 270, 404-410 (1995); Curiel et al., Human Gene Therapy, 3, 147-154 (1992); International Patent Application WO 95/21259).
Due to these desirable properties of adenoviruses, recombinant adenoviral vectors have been used for the cell-targeted transfer of one or more recombinant genes to diseased cells or tissue in need of treatment. In terms of the general structure of an adenovirus, under the electron microscope, an adenovirus particle resembles a space capsule having protruding antennae (Xia et al., Structure, 2, 1259-1270 (1994)). The viral capsid comprises at least six different polypeptides, including 240 copies of the trimeric hexon (i.e., polypeptide II) and 12 copies each of the pentameric penton (polypeptide III) base and trimeric fiber (Xia et al., supra).
An adenovirus uses two separate cellular receptors, both of which must be present, to attach to and infect a cell (Wickham et al., Cell, 73, 309-319 (1993)). First, the adenovirus fiber protein attaches the virus to a cell by binding to an as yet unidentified receptor. Then, the penton base binds to xcex1v integrins, which are a family of heterodimeric cell-surface receptors that mediate cellular adhesion to the extracellular matrix molecules, as well as other molecules (Hynes, Cell, 69, 11-25 (1992)). Once an adenovirus is attached to a cell, it undergoes receptor-mediated internalization into clathrin-coated endocytic vesicles and is stepwise stripped down to the viral double-stranded genome, and then the genome (and some accompanying viral components) subsequently is transported to the cell nucleus, thus initiating infection (Svennson et al., J. Virol., 51, 687-694 (1984); Chardonnet et al., Virology, 40, 462-477 (1970); Greber et al., Cell, 75, 477-486 (1993); Fitzgerald et al., Cell, 32, 607-617 (1983)).
The fiber monomer consists of an amino terminal tail (which attaches noncovalently to the penton base), a shaft (whose length varies among different virus serotypes), and a carboxy terminal globular knob domain (which is necessary and sufficient for host cell binding) (Devaux et al., J. Molec. Biol., 215, 567-588 (1990); xia et al., supra; Green et al., EMBO J., 2, 1357-1365 (1983); Henry et al., J. Virology, 68(8), 5239-5246 (1994)). The regions necessary for trimerization of fiber (which is required for penton base binding) also are located in the knob region of the protein (Henry et al. (1994), supra; Novelli et al., Virology, 185, 365-376 (1991)). The fiber, together with the hexon, determine the serotype specificity of the virus, and also comprise the main antigenic determinants of the virus (Watson et al., J. Gen. Virol., 69, 525-535 (1988)).
This ability of adenoviral fiber and hexon protein to act as targets for a host immune response initially hampered attempts at adenoviral-mediated gene therapy. Namely, alterations in gene expression mediated by adenovirus are not permanent since the vector is not stably maintained. However, following adenoviral vector re-administration to prolong the therapeutic response, neutralizing antibodies can be raised against the adenoviral fiber and/or hexon proteins, thus circumventing protein production (Wohlfart, J. Virology, 62, 2321-2328 (1988); Wohlfart et al., J. Virology, 56, 896-903 (1985)). Fortunately, such an immune response will not be generated with all uses of adenoviral vectors. Similarly, it is now known that if the presence of such neutralizing antibodies impedes adenoviral-mediated intracellular delivery, another adenoviral vector, e.g., another serotype adenoviral vector, or another adenovirus vector lacking the epitope against which the antibody is directed, can be employed instead (Crompton et al., J. Gen. Virol., 75, 133-139 (1994)). Moreover, newer and effective techniques are constantly emerging to prevent an antibody response against the virus from precluding effective re-administration of an adenoviral vector (see, e.g., International Patent Application WO 96/12406; Mastrangeli et al., Human Gene Therapy, 7, 79-87 (1996)).
Thus, adenoviral-mediated gene therapy continues to hold great promise, in particular, with respect to redirecting adenovirus tropism. Namely, even though adenovirus can enter an impressive variety of cell types (see, e.g., Rosenfeld et al., Cell, 68, 143-155 (1992); Quantin et al., Proc. Natl. Acad. Sci., 89, 2581-2584 (1992)); Lemarchand et al, Proc. Natl. Acad. Sci., 89, 6482-6486 (1992); Anton et al., J. Virol., 69, 4600-4606 (1995); LaSalle et al., Science, 259, 988-990 (1993)), there still appear to be cells (e.g., lymphocytes) which are not readily amenable to adenovirus-mediated gene delivery (see, e.g., Grubb et al., Nature, 371, 802-806 (1994); Dupuit et al., Human Gene Therapy, 6, 1185-1193 (1995); Silver et al., Virology 165, 377-387 (1988); Horvath et al., J. Virol., 62(1), 341-345 (1988)). Similarly, even when targeting to cells that readily are infected by adenovirus, in many cases, very high levels of adenovirus particles have been used to achieve transduction. This is disadvantageous inasmuch as any immune response associated with adenoviral infection necessarily would be exacerbated with such high levels.
Accordingly, researchers are seeking new ways to selectively introduce adenoviruses into cells that cannot be infected by adenoviruses, and to increase the effectiveness of adenoviral delivery into cells that are infected by adenoviruses. The general principle of redirecting adenovirus tropism is straightforward. In one common approach, by incorporating peptide binding motifs into an adenovirus coat protein such as fiber protein, the virus can be redirected to bind a cell surface binding site that it normally does not bind (see, e.g., Michael et al., Gene Therapy, 2, 660-668 (1995); International Patent Application WO 95/26412; International Patent Application WO 94/10323; International Patent Application WO 95/05201). A peptide binding motif is a short sequence of amino acids such as an epitope for an antibody (e.g., a bispecific antibody), or a ligand for a cell surface binding site (e.g., a receptor), that can be employed in cell targeting. When the peptide motif binds, for instance to its corresponding cell surface binding site to which adenovirus normally does not bind, or binds with only low affinity, the adenovirus carrying the peptide motif then can selectively deliver genes to the cell comprising this binding site in a specific and/or more efficient manner.
However, simply incorporating a known peptide motif into the fiber protein of an adenovirus may not be enough to allow the virus to bind and effectively transduce a target cell. The effectiveness of the peptide motif in redirecting virus binding to a new cell surface binding site depends on multiple factors, including the availability of the peptide motif to bind to the cell surface receptor, the affinity of the peptide motif for the cell surface binding site, and the number of target binding sites (e.g., receptors) present on the cell targeted for gene delivery. While the lattermost factor currently cannot be manipulated, in in vivo applications, the former two would appear to present areas for improvement of prevailing adenoviral-mediated gene therapy. For instance, earlier researchers have not considered that if the peptide motif is buried within the structure of the fiber protein, and/or masked by the surrounding structure of the protein, the peptide motif will not be able to interact with and bind its target. Similarly, previous researchers have not addressed that it is the affinity of the peptide motif for the cell surface binding site (e.g., receptor) which determines how efficiently the virus can initiate and maintain a binding contact with the target receptor, resulting in cell infection/transduction.
Thus, there remains a need for improved methods of cell targeting, and adenoviral vectors by which this can be accomplished. The present invention seeks to overcome at least some of the aforesaid problems of recombinant adenoviral gene therapy. In particular, it is an object of the present invention to provide improved vectors and methods for cell targeting through provision of a chimeric adenovirus fiber protein comprising a constrained peptide motif. These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the following detailed description.
The present invention provides a chimeric adenoviral fiber protein which differs from the wild-type (i.e., native) fiber protein by the introduction of a nonnative amino acid sequence in a conformationally-restrained (i.e., constrained) manner. The introduction results in the insertion of, or creation of, a constrained peptide motif that confers upon the resultant chimeric adenovirus fiber protein an ability to direct entry into cells of a vector comprising the chimeric fiber protein that is more efficient than entry into cells of a vector that is identical except for comprising a wild-type adenovirus fiber protein, and/or an ability to direct entry into cells that adenovirus comprising the wild-type fiber protein typically does not infect/transduce. The present invention also provides vectors that comprise the chimeric adenovirus fiber protein, and methods of constructing and using such vectors.